The present invention relates to an improved method for labeling antibodies and/or antibody fragments with radionuclides of technetium or rhenium to obtain stable labeling.
Antibodies and/or antibody fragments which are labeled with radionuclides have been shown to be useful for radioimmunoassays, radioimmunodetection of tumors and tumor therapy. In the latter, tumor-related applications as disclosed, e.g., in patents to Goldenberg, U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544 and 4,444,744, one of the preferred radionuclides used for antibody labeling was I-131, because of its convenient half life and the relative ease with which it can be introduced into immunoglobulin molecules and/or fragments thereof. Radioisotopes of iodine have been available for a long time, but have the disadvantage that only a few radioisotopes have appropriate half lives and energies for external imaging, especially imaging using gamma scintigraphic devices. Another disadvantage of using radioiodine for antibody labeling is the in vivo instability of the label, a significant amount of the radioisotope being excreted as elemental iodine within a relatively short time after injection of a radioiodinated antibody into the bloodstream.
Other radioisotopes which are readily available, and which also have convenient half lives and emission energies for gamma imaging, include indium-111 (.sup.111 In) and technetium-99m (.sup.99m Tc). In order to bind .sup.111 In to immunoglobulin molecules or fragments, it is necessary to conjugate a metal chelating agent to the protein since .sup.111 In itself does not bind to immunoglobulin protein. Hnatowich et al, J. Imm. Methods, 65, 147-157 (1983), disclose a method for binding .sup.111 In to antibodies by conjugating diethylenetriaminepentaacetic acid (DTPA) to antibodies, followed by incubation of the DTPA-conjugated antibody with .sup.111 In salts.
Technetium can be bound directly to antibody protein, e.g., as disclosed by Pettit et al, J. Nucl. Med., 21, 59-62 (1980); and Crockford et al, U.S. Pat. No. 4,323,546, or indirectly using DTPA-conjugated antibody, as disclosed in, e.g., Khaw et al, J. Nucl. Med., 23, 1011-1019 (1982). However, several problems are associated with both direct and indirect .sup.99m Tc-labeling of immunoglobulin protein. On the one hand, antibodies which are directly labeled with .sup.99m Tc have been reported to be unstable in vivo, i.e., a significant proportion of the radionuclide dissociates from the labeled antibody fairly quickly upon injection of the labeled antibody into the bloodstream. When labeled antibody is used for external imaging, this instability leads to accumulation of radioactivity in locations other than those at which the radiolabeled antibody localizes. This reduces the resolution of the method by attenuating the localized radioactivity and by increasing the background activity due to non-specific distribution of the radioisotope. Rhodes et al, Tumor Imaging, chapter 12, pages 111-124 (Masson Publ., USA, 1982), disclose that unstable antibodies directly labeled with .sup.99m Tc could be purified using an elaborate permeation chromatographic method. Khaw et al, supra, disclose some loss in antibody activity upon labeling DTPA-conjugated anti-cardiac myosin Fab fragments with .sup.99m Tc, as well as significant liver activity in a scintigram taken 12 hours after injection of the labeled antibody into dogs in which were induced experimental myocardial infarctions. The liver activity was tentatively attributed to incomplete clearance of colloids. No specific determination was made of the percentage of stable label.
Other difficulties reported by earlier workers for .sup.99m Tc labeling of antibodies are reoxidation of reduced technetium (Tc.sup.+3 and/or Tc.sup.+4) to pertechnetate (TcO.sub.4 -) and hydrolysis of reduced technetium ions to form radiocolloids. Residual pertechnetate is difficult to remove as are the colloids, and both tend to contribute to undesirable non-specific background radiation.